Compositions and methods for modulating toll-like receptor 2 activation

ABSTRACT

A method of promoting hair growth of a subject includes administering to follicle cells of the subject a therapeutically effective amount of a TLR2 agonist that promotes TLR2 activation and hair growth of the subject.

RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.13/709,918, filed on Dec. 10, 2012, which is a Continuation ofPCT/US2011/039562, filed Jun. 8, 2011, which claims priority from U.S.Provisional Application No. 61/352,651, filed Jun. 8, 2010, the subjectmatter of which are incorporated herein by reference in their entirety.

GOVERNMENT FUNDING

This invention was made with government support under HL071625 andHL145536 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

This application generally relates to compositions and methods formodulating toll-like receptor 2 (TLR2) activation, and more particularlyto TLR2 agonists and antagonists for modulating TLR2 activation inendothelial and/or immune cells.

BACKGROUND OF THE INVENTION

The process of angiogenesis, or new blood vessel growth, may promotehost defense and tissue repair or exacerbate disease conditions leadingto organ dysfunction. In a number of pathologies, angiogenesis forms astrong reciprocal relationship with the process of inflammation.Recruited inflammatory cells facilitate neovascularization through therelease of proangiogenic growth factors, including vascular endothelialgrowth factor. Newly formed blood vessels promote additional recruitmentof inflammatory cells, thereby promoting the chronic aspect ofinflammation. Various types of inflammatory cells, in particular ofmyeloid origin, are guided by and contribute to hyperoxidativeconditions characterized by the presence of oxidized lipids and modifiedproteins.

Hyperoxidative conditions lead to the generation of a host of oxidativeproducts, including hydroxy-ω-oxoalkenoic acids and their esters. Whenpresent in oxidized phospholipids, these molecules are recognized byscavenger receptor CD36 and contribute to atherosclerosis progressionand platelet hyper-reactivity. Hydrolysis followed by reaction of theresulting unesterified hydroxy-ω-oxoalkenoic acids with proteins, orreaction of the esterified hydroxy-ω-oxoalkenoic acids with proteinsfollowed by hydrolysis gives rise to a family of carboxyalkylpyroleprotein adducts, among them 2-(ω-carboxyethyl) pyrrole adducts andsimilarly modified compounds. These adducts, which are present inoxidized LDL, can accumulate in atherosclerotic plaques. They are alsofound in the retina in photoreceptor outer segments and retinalpigmented endothelial cells, where they contribute to age-relatedmacular degeneration progression, inter alia, by promoting choroidalneovascularization.

SUMMARY OF THE INVENTION

This application generally relates to compositions and methods formodulating toll-like receptor 2 (TLR2) activation, and more particularlyto TLR2 agonists and antagonists for modulating TLR2 activation in cellsexpressing TLR2, such as endothelial cells and/or immune cells.

One aspect of the application relates to a method of modulatingangiogenesis in a tissue of a subject. The method can includeadministering to tissue of the subject that includes endothelial cells atherapeutically effective amount of an agent that promotes or inhibitsTLR2 activation.

Another aspect of the application relates to a topical or localformulation for treating a wound. The topical or local formulation caninclude a therapeutically effective amount of a TLR2 agonist thatpromotes TLR2 activation and at least one carrier. The topicalformulation when administered to a wound of a subject can promote woundhealing and/or accelerate wound closure.

A further aspect of the application relates to a pharmaceuticalcomposition for treating a neoplastic disorder in a subject. Thepharmaceutical composition can include an agent that inhibits complexingof TLR2 with carboxylalkyl pyrrole (CAP) adducts in endothelial cells,which are in or proximate neoplastic tissue of the subject.

Yet another aspect of the application relates to a method for promotinghair growth in a subject. The method can include administering tofollicle cells of the subject a therapeutically effective amount of aTLR2 agonist that promotes TLR2 activation and hair growth of thesubject.

Another aspect of the application relates to a topical formulation forpromoting hair growth in a subject. The topical formulation can includea therapeutically effective amount of a TLR2 agonist that promotes TLR2activation and at least one carrier. The topical formulation whenadministered to follicle cells of the subject can promote hair growth.

A further aspect of the application relates to a method of modulating ina subject one or more inflammatory and/or autoimmune diseases ordisorders mediated by TLR2. The method can include administering to thesubject a composition comprising an agent that modulates one or more CAPadducts in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the present invention will becomeapparent to those skilled in the art to which the present inventionrelates upon reading the following description with reference to theaccompanying drawings, in which:

FIGS. 1A-G are series of immunostains showing that products of lipidoxidation represented by 2-(ω-carboxyethyl) pyrolle (CEP) areaccumulated in wounded areas and present at high levels in tumors and inaging tissues. In FIG. 1A, co-staining for CEP and CD31 in normal skin(control) and wounded tissue collected 5 (5d) and 28 days (28d) afterinjury is shown (scale bar is 10 μm). Quantification of CEP staining ison the right. Bars represent fold increase over control (skin prior toinjury)±s.e.m., n=4. In FIG. 1B, CEP is present in bone marrow derivedcells. Wound tissue was collected 5 days after injury from micetransplanted with bone marrow from DsRed expressing mice (scale bar is10 μm). FIG. 1C shows co-staining for Gr-1, F4/80 and endogenous CEP inwound tissue before injury (0), 5 and 28 days after injury is indicated(scale bar is 10 μm). FIG. 1D shows that CEP is accumulated in highlyvascularized tissues. Co-staining for CEP and CD31 in implanted mouseB16 melanoma and normal skin of C57B1/6 mice (WT) is shown (scale bar is10 μm). Quantifications of CEP staining intensity and vascularizationbased on CD31 staining are shown on the right. Bars represent foldincrease over control (normal skin)±s.e.m., n=5. FIG. 1E shows that CEPand CD31 staining of human melanoma and normal skin are from the samesubjects (scale bar is 10 μm). Quantifications of CEP staining intensityand vascularization (CD31) are shown on the right. Bars represent foldincrease over control (normal skin)±s.e.m., n=5. FIG. 1F shows thatendogenous CEP in normal skeletal muscle tissue is present within thevessel wall. Co-staining for CEP and CD31-top; CEP and SMA-bottom (scalebar is 10 μm). FIG. 1G shows that CEP is accumulated in aging tissues.Tissue from 5 weeks (young), and 11 months (old) C57B1/6 mice wereimmunostained with anti-CEP and anti-CD31 antibodies (scale bar is 10μm). Quantifications of CEP staining intensity is shown on the right.Bars represent fold increase over control (tissue from youngmice)±s.e.m., n=4;

FIGS. 2A-H are a series of micrographs showing that the pro-angiogeniceffect of oxidized adducts is not restricted to a particular type ofendothelial cell and is independent of CD36 and SR-B1 expression. FIG.2A shows a HUVEC tube formation assay: left-representative micrographsof control (no treatment) and CEP-mouse serum albumin (MSA),CEP-dipeptide and 2-(ω-carboxypropyl) pyrrole (CPP)-human serum albumin(HAS) treated cells (scale bar is 60 μm); right-quantification of tubeformation assay in the presence of CEP-MSA, CEP-dipeptide, CPP-HSA orVEGF (positive control) as indicated. Bars represent fold increase overcontrol±s.e.m., n=4. FIG. 2B shows a Mouse Lung Microvascular EC (MLEC)tube formation assay: left-representative micrographs of control (notreatments) and CEP-MSA, CEP-dipeptide and CPP-HSA treated cells (scalebar is 60 μm); right-quantification of tube formation assay (VEGF aspositive control) as indicated. Bars represent fold increase overcontrol±s.e.m., n=4. FIG. 2C shows a Mouse aortic ring assay:left-representative micrographs of control (no treatments) and CEP-MSA,CEP-dipeptide and CPP-HSA treated cells; right-numbers of microvesselsper aortic ring±s.e.m. are shown at different time points after additionof CEP-MSA, CEP-dipeptide, CPP-HSA or VEGF (positive control) asindicated, n=5. FIG. 2D shows a HUVEC migration assay: cells weretreated with CEP-MSA, CEP-dipeptide, CPP-HSA or VEGF at the indicatedconcentration or remained untreated (control). Bars represent percentincrease over control±s.e.m., n=4. FIGS. 2E-H show aortic ring (FIG. 2Eand FIG. 2G) and tube formation (FIGS. 2F and 2H) assays usingCD36^(−/−) (FIGS. 2E and 2F) and SR-B1^(−/−) (FIGS. 2G and 2H) mice;left-representative micrographs of control (no treatments) and CEP-MSA,CEP-dipeptide, CPP-HSA treated cells (scale bar is 60 μm);right-quantification of sprouts stimulated by CEP-MSA, CEP-dipeptide,CPP-HSA or VEGF (positive control) was performed as described in theExample below (bars represent fold increase over control±s.e.m., n=5);

FIGS. 3A-F shows that angiogenesis induced by oxidized lipid products ismediated by toll-like receptor 2 (TLR2). In FIG. 3A, HUVEC werestimulated with CEP or remained unstimulated. Tube formation assay wasperformed in the presence of blocking antibodies to TLR2 and TLR4 ornon-immune isotype matched to IgG as indicated. Bars represent percentchange over control±s.e.m., n=4. FIG. 3B shows that oxidized adductsfail to induce angiogenesis in MLEC tube formation assay usingTLR2^(−/−) mice. Left-representative micrographs of control (notreatments), CEP-MSA, CEP-dipeptide and CPP-HSA treated cells, scale baris 60 μm; right-bars represent fold increase in vascularization overcontrol after stimulation with CEP-MSA, CEP-dipeptide, CPP-HSA or VEGF(positive control) as indicated, ±s.e.m., n=4. FIG. 3C shows thatTLR2^(−/−) but not TLR2^(−/−) cells respond to stimulation withCEP-dipeptide in aortic ring assay. Left-representative micrographs ofcontrol (no treatments), CEP-dipeptide and VEGF treated aortic ringsfrom TLR2^(−/−) mice, scale bar is 60 μm; right-bars represent numbersof microvessels per ring±s.e.m. after stimulation of TLR2^(+/+) andTLR2^(−/−) cells with CEP-dipeptide or VEGF as indicated, n=4. FIG. 3Dshows that TLR2 ligand Pam3CSK4 induces angiogenesis in TLR2^(+/+) butnot TLR2^(−/−) cells. Assay and quantifications are performed as inFIGS. 3C, 3E and 3F. CEP promotes angiogenesis in hind limb ischemiamodel using TLR2^(+/+) but not TLR2^(−/−) mice. Surgery, CEP injections,and tissue collection were performed as described in the Example below.In FIG. 3E, immunostaining for SMA is shown in PBS and CEP-treatedtissues; scale bar is 40 μm. Bars represent vascular density andvascular area±s.e.m. after treatment with CEP or PBS (control) asindicated, n=6. FIG. 3F shows LDI images of hind limb blood flow aftertreatment with CEP or PBS (control) as indicated are shown before,immediately after and 5 days post-hind limb ischemia (HLI) surgery. Theperfusion ratio calculated as described in the Example below±s.e.m. areshown, n=8 (days after surgery are indicated);

FIGS. 4A-E show wound angiogenesis in TLR2^(+/+) and TLR2^(−/−) micebefore and after bone marrow transplantation, and the role of Myd88 andRac1. FIG. 4A shows that CEP promotes wound healing and vascularizationin TLR2^(+/+) but not TLR2^(−/−) mice. Images of skin inner flap at 7days after injury are shown. FIGS. 4B-C show that the response to CEPdoes not depend on TLR2 in bone-marrow derived cells. TLR2^(−/−) andTLR2^(+/+) mice were transplanted with TLR2^(+/+) bone marrow asindicated and a wound assay was performed as in the Example below. FIG.4B shows representative images of wounds are shown; scale bar is 1 mm.Right-bars represent reductions in wound area±s.e.m. upon treatment withCEP or PBS, n=5. FIG. 4C shows representative images of tissues stainedfor CD31 and CEP. Bottom: (i) bars represent fold changes in vasculararea over control (PBS-treated)±s.e.m. upon treatment withCEP-dipeptide, n=5; and (ii) fold changes in vascular density overcontrol (PBS-treated)±s.e.m. upon treatment with CEP dipeptide, n=5.FIG. 4D shows that CEP does not induce angiogenic response inMyd88^(−/−) mice. Aortic ring assay was performed using MyD88^(+/+) andMyD88^(−/−) mice in the presence of CEP or VEGF as indicated; (i)representative micrographs; and (ii) bars represent numbers ofmicrovessels per ring)±s.e.m., n=5. FIG. 4E shows that CEP treatmentpromotes Rac1 activation in WT but not in TLR2^(−/−) and MyD88^(−/−)cells: (i) levels of Rac1GTP and total Rac1 in lysates of EC treatedwith or without CEP are shown; and (ii) bars represent fold increase inRac1GTP calculated by densitometry analysis;

FIG. 5 is a schematic diagram of CAP protein adduct biogenesis;

FIGS. 6A-B show that the CEP effect is integrin-dependent andVEGFR2-independent. For the cell adhesion assay, staining was done withhematoxylin (scale bar 40 μm, right-average cell number calculated insix independent fields±s.e.m., RGD—integrin ligand competitor)(**P<0.01, ***P<0.001). In FIG. 6B, HUVEC cells were treated with VEGFor CEP-dipeptide for indicated period of time, lysed, and probed forphosphor-VEGFR2 (top) or total actin (bottom);

FIGS. 7A-D are a series of graphs showing that: CEP-induced responsesare TLR2 dependent (FIG. 7A); migration of MLEC cells in response to theindicated protein adducts is dependent on TLR2 expression (FIG. 7B);HUVEC tube formation in response to Pam₃CSK₄ is inhibited by TLR2blocking antibodies but not isotype control antibodies (FIG. 7C); andHUVEC response to Pam₃CSK₄ is TLR2 dependent. In FIG. 7A, HUVECmigration in the presence of TLR2 or TLR4 blocking antibodies isindicated (bars represent average cell number in six fields±s.e.m.)(NS—not significant, ***P<0.001). In FIG. 7B, cell migration as % tocontrol sample±s.e.m. is shown (*P<0.05, ***P<0.001). In FIG. 7C, CEPand VEGF-induced responses are shown for comparison (scale bar 60 μm;right-calculated average tube length±s.e.m., n=4) (NS—not significant,***P<0.001). In FIG. 7D, the effects of CEP and VEGF are shown forcomparison (fold increase over control±s.e.m., n=4) (NS—not significant,*P<0.05);

FIG. 8 is a series of images of a ligated femoral artery (post-operationday 28, CEP or vehicle only intramuscular injection as indicated) (scalebar 150 μm; right-magnified view of the area indicated on the left);

FIGS. 9A-B are a series of plots showing TLR2 recombinant proteinbinding to CEP-KLH adduct over a range of TLR2 protein concentrations(FIG. 9A) (KLH protein is shown for comparison; optical density±s.e.m.,n=3), and Luciferase reporting assay for NF-κB activation (FIG. 9B)(TLR2 or empty vector transfection shown on bottom) (Luciferasecounts±s.e.m., n=3, ***P<0.001);

FIGS. 10A-B show tumor vasculature (melanoma) in TLR2^(−/−) mice with WTbone marrow compared to WT mice transplanted with TLR2^(−/−) bonemarrow. FIG. 10A is a series of immunohistochemistry images showingvasculature in excised tumors from WT mice with WQT bone marrow,TLR2^(−/−) mice with WT bone marrow (middle), and WT mice withTLR2^(−/−) bone marrow (CD31 staining as an endothelial marker is shownin red) (NS—not significant);

FIG. 11 is a series of images showing a wound assay with whitepetrolatum CEP-dipeptide added topically (note increased vascularizationon the right, CEP application);

FIGS. 12A-B illustrates (A) images of WT and TLR2 null mice treated withpetroleum with or without CEP and (B) images of H&E stained sections ofskin showing hair follicles of WT and TLR2 null mice treated with orwithout CEP.

FIG. 13 illustrates a chart showing quantitative results of increaseddensity of hair follicles upon CEP application in TLR2+/+ but notTLR2−/− mice.

FIGS. 14A-B illustrates (A) images of bone marrow chimeras of WT(TLR2^(+/+)) and null (TLR2^(−/−)) mice treated with petroleum with orwithout CEP and (B) images of H&E stained sections of skin showing hairfollicles of bone marrow chimeras of WT and TLR2 null mice treated withor without CEP.

FIG. 15 illustrates a chart showing quantitative results of increaseddensity of hair follicles upon CEP application in bone marrow chimerasof TLR2^(+/+) but not TLR2^(−/−) mice.

DETAILED DESCRIPTION

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of theapplication.

As used herein, the term “neoplastic disorder” can refer to a diseasestate in a subject in which there are cells and/or tissues whichproliferate abnormally. Neoplastic disorders can include, but are notlimited to, cancers, sarcomas, tumors, leukemias, lymphomas, and thelike.

As used herein, the term “neoplastic cell” can refer to a cell thatshows aberrant cell growth, such as increased, uncontrolled cell growth.A neoplastic cell can be a hyperplastic cell, a cell from a cell linethat shows a lack of contact inhibition when grown in vitro, a tumorcell, or a cancer cell that is capable of metastasis in vivo.Alternatively, a neoplastic cell can be termed a “cancer cell” or “tumorcell”. Non-limiting examples of cancer cells can include melanoma,breast cancer, ovarian cancer, prostate cancer, sarcoma, leukemicretinoblastoma, hepatoma, myeloma, glioma, mesothelioma, carcinoma,leukemia, lymphoma, Hodgkin lymphoma, Non-Hodgkin lymphoma,promyelocytic leukemia, lymphoblastoma, thymoma, lymphoma cells,melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells,hepatoma cells, myeloma cells, glioma cells, mesothelioma cells, andcarcinoma cells.

As used herein, the term “tumor” can refer to an abnormal mass orpopulation of cells that result from excessive cell division, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

As used herein, the terms “treating” or “treatment” of a disease ordisorder can refer not only to ameliorating symptoms associated with thedisease or disorder, but also preventing or delaying the onset of thedisease or disorder, and/or lessening the severity or frequency ofsymptoms of the disease or disorder. Thus, the terms “treating” or“treatment” can include complete or incomplete eradication of thedisease or disorder.

As used herein, the term “polymer” can refer to a molecule formed by thechemical union of two or more chemical units. The chemical units may belinked together by covalent linkages. The two or more combining units ina polymer can be all the same, in which case the polymer may be referredto as a homopolymer. The chemical units can also be different and, thus,a polymer may be a combination of the different units. Such polymers maybe referred to as copolymers.

As used herein, the term “subject” can refer to any animal, including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish), non-human primates, ovines, bovines,ruminants, lagomorphs, porcines, caprines, equines, canines, felines,ayes, etc.), which is to be the recipient of a particular diagnosticand/or therapeutic application.

As used herein, the term “agonist” can refer to a substance that bindsto a receptor of a cell and induces a response. An agonist often mimicsthe action of a naturally occurring substance, such as a ligand.

As used herein, the term “antagonist” can refer to a substance thatattenuates the effects of an agonist.

As used herein, the terms “co-administration” or “co-administered” canrefer to the administration of at least two different substancessufficiently close in time to modulate a physiological response (e.g.,an immune response). Co-administration can refer to simultaneousadministration, as well as temporally spaced order of up to several daysapart and of at least two different substances in any order, either in asingle dose or separate doses.

As used herein, the term “polypeptide” can refer to a molecule composedof monomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term can indicate a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,dipeptides, tripeptides, oligopeptides, and proteins can be includedwithin the definition of polypeptide. This term can also refer to theproducts of post-expression modifications of a polypeptide, such asglycosylation, hyperglycosylation, acetylation, phosphorylation, and thelike. A polypeptide may be derived from a natural biological source orproduced by recombinant technology, and is not necessarily translatedfrom a designated nucleic acid sequence. A polypeptide may be generatedby any manner known in the art, such as chemical synthesis.

As used herein, the terms “therapeutically effective amount” or“pharmaceutically effective amount” can refer to an amount of asubstance and/or composition sufficient to affect a desired biologicaleffect, such as a beneficial result, including, without limitation,prevention, diminution, amelioration or elimination of signs or symptomsof a disease or disorder. The total amount of each active component of apharmaceutical composition or method may be sufficient to show ameaningful subject benefit (e.g., promoting wound healing). A“pharmaceutically effective amount” will depend upon the context inwhich it is being administered. A pharmaceutically effective amount maybe administered in one or more prophylactic or therapeuticadministrations. When applied to an individual active ingredient,administered alone, the terms can refer to that ingredient alone. Whenapplied to a combination, the terms can refer to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

As used herein, the term “modulate” can refer to a change in thebiological activity of a biologically active molecule, such as a cellreceptor. Modulation can be an increase or a decrease in activity, achange in binding characteristics, or any other change in thebiological, functional, or immunological properties of biologicallyactive molecules.

This application relates to compositions and methods for modulatingtoll-like receptor 2 (TLR2) activation, and more particularly to TLR2agonists and antagonists for modulating TLR2 activation in cellsexpressing TLR2, such as endothelial and/or immune cells. Although it isnot necessary to understand the mechanisms in order to practice themethods or compositions described herein, and it is not intended thatthe application be so limited, it is shown herein that certain endproducts of lipid oxidation, such as ω-(2-carboxyalkyl)pyrrole (CAP)adducts and similar compounds are generated during inflammation andwound healing and accumulate at high levels in aging tissues and highlyvascularized tumors. Carboxyalkyl pyrrole (CAP) adducts are recognizedby TLR2 (but not TLR4) and lead to a VEGF-independent angiogenicresponse. Lipid oxidation products (e.g., CAP adducts) were found topromote angiogenic responses of endothelial cells by activating the TLR2signaling pathway in a MyD88-dependent manner, leading to Rac1activation which, in turn, facilitates integrin function.

CAP adducts if provided exogenously, as shown in the Examples, can treathind limb ischemia, wound, and hair loss through TLR2 in aMyd88-dependent manner. Moreover, TLR2 antagonists if providedexogenously can substantially inhibit aberrant angiogenesis associatedCAP adduct activation. This application therefore provides compositionsand methods for modulating TLR2 activation in endothelial and/or immunecells to treat a variety of conditions, such as ischemia, wounds,dermatological disorders, cancer, autoimmune diseases, and inflammatorydiseases.

One aspect of the application can include a method for modulatingangiogenesis in a tissue of subject by administering to the tissue ofthe subject that includes at least one endothelial cell atherapeutically effective amount of an agent that promotes or inhibitsTLR2 activation. Angiogenesis is a physiological process involving thegrowth of new blood vessels from pre-existing vessels.

In an aspect of the application, an agent that promotes angiogenesis inthe tissue can include a TLR2 agonist. Generally, TLR2 agonists caninclude any substance or molecule that binds to or activates TLR2present on cells (e.g., endothelial, endothelial precursor cells, and/orimmune cells) and induces a response in the cells. For example, TLR2agonists that bind to TLR2 can promote TLR2:Myd88 complex formation,lead to or increase binding of Rac1 with GTP, and/or promote integrinexpression. Moreover, TLR2 agonists that bind to TLR2 can include anysubstance or molecule that binds to TLR2 and promotes migration ofendothelial cells and/or endothelial precursor cells from a parentvessel wall into the surrounding matrix to a site of angiogenesis. Bybinding to or activating TLR2 in an endothelial cell, endothelialprecursor cell, and/or immune cell, the TLR2 agonist can increase orpromote angiogenesis in a subject.

One example of a TLR2 agonist can include a pyrrole adduct thatpreferentially binds TLR2 but not TLR4. In some embodiments, the pyrroleadduct can include CAP adducts, such as 2-(ω-carboxyheptyl)pyrrole(CHP), 2-(ω-carboxypropyl)pyrrole (CPP), and 2-(ω-carboxyethyl)pyrrole(CEP) adducts, as well as other pyrrole adducts that are generated fromoxidation of poly-unsaturated fatty acids (PUFAs). For example,oxidative fragmentation of linoleic acid or 5-hydroxy-8-oxooct-6-enoicacid (HOOA) can produce 9-hydroxy-12-oxododec-10-enoic acid (HODA),respectively, which reacts with protein to generate CHP or CPP adducts,respectively. CHP and CPP protein adducts can arise not only fromlinoleic or arachidonic acid, but also from oxidation of other commonPUFAs. For example, CEP protein adducts are a unique proteinmodification, which are derived from the oxidation of docosahexaenoate(DHA)-containing lipids.

In other embodiments of the application, pyrrole adducts can have thegeneral formula X-A, wherein A is a substituted pyrrole and X is apeptide, dipeptide, polypeptide, small molecule, or polymer thatincludes an amine group that binds to the amine of the substitutedpyrrole, or pharmaceutically acceptable salts thereof. For example, thesubstituted pyrrole adduct can have the following general formula:

wherein X is a peptide, dipeptide, polypeptide, small molecule, orpolymer that includes an amine group that binds to the amine of thesubstituted pyrrole, and R is a hydrophilic, hydropobic, or lipophilicgroup, or pharmaceutically acceptable salts thereof.

In another example of the present invention, the substituted pyrroleadduct can have the following general formula:

wherein X is a peptide, dipeptide, polypeptide, small molecule, orpolymer that includes an amine group that binds to the amine of thesubstituted pyrrole, and n is an integer from 1-12, or pharmaceuticallyacceptable salts thereof.

In other embodiments, the substituted pyrrole adduct can include a CEPadduct, a CHP adduct, a CPP adduct, and/or combinations thereof.

It will be appreciated that TLR2 agonists can include other moleculesbesides substituted pyrroles, such as naturally occurring and/orsynthetic peptides. One example of a synthetic peptide that may be usedas a TLR2 agonist is Pam₃CSK₄. Pam₃CSK₄ is a synthetic tripalmitoylatedlipopeptide that activates the acylated amino terminus of bacteriallipoproteins. It also activates monocytes and macrophages and is apotent activator of NF-κB. Pam₃CSK₄ is recognized by a heterodimerformed between TLR1 and TLR2.

Upon administering a therapeutically effective amount of a TLR2 agonistto the tissue of a subject containing endothelial cells, the TLR2agonist can bind to TLR2 and thereby activate the TLR2 signaling pathwayin a MyD88-dependent manner Activated MyD88 can then lead to Rac1activation and integrin expression. Activated endothelial cells canbegin to release enzymes (e.g., proteases) that degrade the basementmembrane and allow the endothelial cells to escape from the original(parent) vessel walls. The endothelial cells can then proliferate intothe surrounding matrix and form solid sprouts connecting neighboringvessels. As sprouts extend toward the source of the angiogenic stimulus,the endothelial cells can migrate in tandem using the integrins. Thesesprouts can then form loops to become a full-fledged vessel lumen as theendothelial cells migrate to the site of angiogenesis.

The stimulation of angiogenesis can play an important role in a varietyof physiological processes, such as embryonic development, woundhealing, organ regeneration and female reproductive processes, such asfollicle development in the corpus luteum during ovulation and placentalgrowth after pregnancy. Additionally, millions of patients per year inthe U.S. suffer from myocardial infarction (MI) and/or critical limbischemia. Many millions more suffer from related syndromes due toatherosclerosis. Many of these patients will benefit from the ability tostimulate angiogenesis in ischemic areas.

Where a composition comprising the TLR2 agonist is to be used fortherapeutic purposes, the dose(s) and route of administration willdepend upon the nature of the patient and condition to be treated, andwill be at the discretion of the attending physician or veterinarian.Examples of routes that can be used for the administration of the TLR2agonist include oral, subcutaneous, intramuscular, intraperitoneal orintravenous injection, parenteral, topical application, implants etc.

In one example, a therapeutically effective amount of a pharmaceuticalcomposition comprising the TLR2 agonist can be administered to a woundor an area proximate a wound in a subject to promote healing of thewound. The terms “promoting wound healing” or “promoting healing ofwound” can refer to the augmenting, improving, increasing, or inducingclosure, healing, or repair of a wound. Wounds treatable by thepharmaceutical compositions described herein that include a TLR2 agonistcan include any injury to any portion of the body of a subject (e.g.,internal wound or external wound) including: dermal wounds, acuteconditions or wounds, such as thermal burns, chemical burns, radiationburns, burns caused by excess exposure to ultraviolet radiation (e.g.,sunburn); damage to bodily tissues, such as the perineum as a result oflabor and childbirth; injuries sustained during medical procedures, suchas episiotomies; trauma-induced injuries, such as cuts, incisions,excoriations, injuries sustained as result of accidents, ulcers, such aspressure ulcers, diabetic ulcers, plaster ulcers, and decubitus ulcer,post-surgical injuries. Wounds can also include chronic conditions orwounds, such as pressure sores, bedsores, conditions related to diabetesand poor circulation, and all types of acne. In addition, woundstreatable by the present invention can include dermatitis, such asimpetigo, intertrigo, folliculitis and eczema, wounds following dentalsurgery, periodontal disease, and tumor associated wounds.

The TLR2 agonist can be formulated for either topical and/or localdelivery, depending upon the type and severity of the wound. The term“local delivery” can refer to delivery of a pharmaceutical ortherapeutic composition to a defined area or region of the body. Theterm “topical delivery” can refer to delivery of a pharmaceutical ortherapeutic composition to a dermal surface (e.g., epidermal) of thebody. Topical formulations can include those for delivery via the mouth(buccal) and to the skin such that at least one layer of skin (i.e., theepidermis, dermis, and/or subcutaneous layer) is contacted with a TLR2agonist.

Formulations for topical administration can include ointments, creams,gels, and pastes comprising a TLR2 agonist to be administered with apharmaceutically acceptable carrier. Topical formulations can beprepared using oleaginous or water-soluble ointment bases, as is wellknown to those in the art. For example, such formulations may includevegetable oils, animal fats, and more preferably semisolid hydrocarbonsobtained from petroleum. Particular components that may be used caninclude white ointment, yellow ointment, cetyl esters wax, oleic acid,olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starchglycerite, white wax, yellow wax, lanolin, anhydrous lanolin, andglyceryl monostearate. Various water-soluble ointment bases may also beused including, for example, glycol ethers and derivatives, polyethyleneglycols, polyoxyl 40 stearate, and polysorbates.

In one example of the present invention, a TLR2 agonist, such as a CEPadduct can be formulated into a topical formulation (e.g., a cream) forapplication to, and treatment of, a wound.

A pharmaceutical or therapeutic composition comprising a TLR2 agonistcan be applied directly to the wound and/or about the periphery of thewound for a time and at a dosage sufficient to promote angiogenesis atthe wound site and thereby facilitate revascularization and closure ofthe wound. As is well known in the art, dosage for any one subjectdepends on many factors, including the subject's size, body surfacearea, age, the particular composition to be administered, sex, time androute of administration, general health, and other drugs beingadministered concurrently. The specific dosage of a pharmaceutical ortherapeutic composition comprising a TLR2 agonist can be readilydetermined by one skilled in the art.

Upon administration of a pharmaceutical or therapeutic compositioncomprising a TLR2 agonist to a wound, the TLR2 agonist can bind to TLR2on an endothelial cell at and/or proximate to the wound site. Binding ofthe TLR2 agonist to TLR2 can activate the TLR2 signaling pathway in aMyD88-dependent manner and, as described above, lead to Rac1 activationand integrin expression. As activated endothelial cells proliferate intothe surrounding matrix and form solid sprouts connecting neighboringvessels, the wound site can become revascularized and the damaged tissueeventually replaced with normal or healthy tissue.

In another example, a therapeutically effective amount of a TLR2 agonistcan be administered to ischemic tissue of a subject to treat an ischemicdisorder in the subject. Ischemic disorders treatable by the TLR2agonist can include peripheral vascular disorders, pulmonary emboli,venous thromboses, myocardial infarction, transient ischemic attacks,unstable angina, cerebral vascular ischemia, reversible ischemicneurological deficits, ischemic kidney disease, and stroke disorders.Ischemic disorders can also include iatrogenically-induced ischemicdisorders, such as those resulting from a subject undergoing, forexample, angioplasty, heart surgery, lung surgery, spinal surgery, brainsurgery, vascular surgery, abdominal surgery, kidney surgery, or organtransplantation surgery. Examples of organ transplantation can includeheart, lung, pancreas, kidney, and liver translation surgeries.

Ischemic disorders can be treated by administering an amount of a TLR2agonist to ischemic tissue of subject over a period of time sufficientto promote angiogenesis in the ischemic tissue. For example, the periodof time that a TLR2 agonist can be administered to an ischemic tissuemay be from about immediately after onset of the ischemic disorder toabout days, weeks, or months after the onset of the ischemic disorder.

The TLR2 agonist can be administered directly to or about the peripheryof ischemic tissue to promote angiogenesis in, and thusrevascularization of, the ischemic tissue. For example, the TLR2 agonistcan be delivered to or about the periphery of the ischemic tissue byadministering the TLR2 neat or in a pharmaceutical or therapeuticcomposition. The pharmaceutical or therapeutic composition can providelocalized release of the TLR2 to the ischemic tissue (or cells) beingtreated. Pharmaceutical compositions can generally include an amount ofa TLR2 agonist admixed with an acceptable pharmaceutical carrier, suchas a sterile aqueous solution, to give a range of final concentrations,depending on the intended use. The techniques of preparation aregenerally well known in the art as exemplified by Remington'sPharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980,incorporated herein by reference. For human administration, preparationsshould meet sterility, pyrogenicity, general safety, and puritystandards as required by FDA Office of Biological Standards.

By way of example, where the ischemic tissue to be treated is infarctedmyocardium, a TLR2 agonist, such as a CEP adduct can be formulated intoa pharmaceutical or therapeutic composition for direct injection intothe infarcted tissue. For instance, a CEP adduct can be formulated in aunit dosage injectable form (e.g., solution, suspension, and/oremulsion). Examples of pharmaceutical formulations suitable forinjection can include sterile aqueous solutions or dispersions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. Using a syringe, for example, atherapeutically effective amount of the pharmaceutical or therapeuticcomposition comprising the CEP adduct can be injected into and/orproximate the infarcted myocardium of the subject. Upon injection, theCEP adduct can bind to TLR2 on an endothelial cell in and/or proximateto the infarcted myocardium. Binding of the CEP adduct to TLR2 canactivate the TLR2 signaling pathway in a MyD88-dependent manner and, asdescribed above, lead to Rac1 activation and integrin expression.Activated endothelial cells can then proliferate into the surroundingmatrix and form solid sprouts to connect neighboring vessels. Theinfarcted tissue can become revascularized and eventually replaced withnormal or healthy myocardium.

Another aspect of the application can include a method of inhibitingpathological angiogenesis in a subject by administering atherapeutically effective amount of an agent that substantially inhibitscomplexing of TLR2 with CAP adducts in endothelial cells and/or immunecells of a tissue. As used herein, the terms “inhibit”, “inhibiting” or“inhibition” can include any measurable, reproducible, and/orsubstantial reduction in: the interaction of CAP adducts and TLR2;angiogenesis; symptoms of diseases or disorders correlated toangiogenesis; or any other activities that complex formation of CAPadducts and TLR2 may mediate. A substantial reduction can include a“reproducible”, i.e., consistently observed reduction in complexformation and/or angiogenesis.

In one example, an agent that substantially inhibits complexing of TLR2with CAP adducts in endothelial cells of a tissue can include a TLR2antagonist. Generally, TLR2 antagonists can include any substance ormolecule that attenuates the effects of a TLR2 agonist. One example of aTLR2 antagonist can include a TLR2 antibody that binds to TLR2 andprevents or inhibits complex formation between CAP adducts and TLR2.Anti-TLR2 antibodies can include whole antibodies, e.g., of any isotype(IgG, IgA, IgM, IgE, etc) and/or fragments thereof that are specificallyreactive with a TLR2 epitope. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility and/orinteraction with a specific epitope of interest. Thus, antibodies and/orfragments thereof can include segments of proteolytically-cleaved orrecombinantly-prepared portions of an antibody molecule and/or fragmentthereof that is/are capable of reacting with a TLR2 epitope.Non-limiting examples of such proteolytic and/or recombinant fragmentscan include Fab, F(ab′)2, Fab′, Fv, and single chain antibodies (scFv)containing a V[L] and/or V[H] domain joined by a peptide linker. ThescFv's may be covalently or non-covalently linked to form antibodieshaving two or more binding sites. Antibodies and/or fragments thereofcan include polyclonal, monoclonal, or other purified preparations ofantibodies, recombinant antibodies, monovalent antibodies, andmultivalent antibodies. Antibodies and/or fragments thereof may behumanized and further include engineered complexes that compriseantibody-derived binding sites, such as diabodies and triabodies.

In another example, an agent that substantially inhibits complexing ofTLR2 with CAP adducts in endothelial cells of a tissue can include anysubstance or molecule that inhibits the activity and/or formation (e.g.,expression) of a CAP adduct, such as a CEP protein adduct. As usedherein, the terms “inhibit”, “inhibiting” or “inhibition” can includeany measurable, reproducible, and/or substantial reduction in: theformation of CAP adducts; the activity of CAP adducts; the interactionof CAP adducts and TLR2; and/or any other pathological activitiescomplex formation of CAP adducts and TLR2 may mediate. An agent thatinhibits a CAP adduct can alter CAP adduct activity or CAP adductformation by a variety of means. The inhibition can be partial orcomplete inhibition of CAP adduct activity and/or formation. Inaddition, the agent can inhibit the CAP adduct directly (specificallyinteract) or indirectly (non-specifically interact).

For example, the agent can inhibit one or more biological activities ofa CAP adduct (e.g., a CEP protein adduct). An example of a biologicalactivity of a CAP adduct is angiogenesis. The agent can bind to all or aportion of the CAP adduct (e.g., a portion of the CAP adduct, the CAPportion of the CAP adduct, or the adduct portion of the CAP adduct)under conditions in which the angiogenic activity of the CAP adduct isinhibited.

Alternatively, the agent can inhibit formation of a CAP adduct. Theagent can prevent CAP protein adducts from forming by, for example,hydrolyzing CAP protein adducts that have previously formed and therebyregenerating the primary amino group found in the unmodifiedbiomolecule. In one example, the agent can interact with HOHA or itsesters, e.g., phospholipid derivatives containing a HOHA acyl groupesterified to the sn-2 position, and/or the protein which forms anadduct with HOHA prior to formation of a CEP protein adduct, therebypreventing CEP protein adducts from forming. Alternatively, the agentcan interact with an upstream product (e.g., DHA) of the reaction (i.e.,leading to formation of CEP protein adducts to prevent CEP proteinadduct formation) and/or interact with the CEP protein adduct or portionthereof after CEP protein adducts have formed so that the pyrrole moietyof the CEP and the protein of the CEP protein adduct is disrupted (e.g.,the agent can cleave the CEP group from the protein).

Examples of agents that can inhibit receptor-mediated effects of CAPadducts (e.g., CEP protein adducts) can include nucleic acids, fragmentsor derivatives thereof and vectors comprising such nucleic acids (e.g.,a nucleic acid molecule, cDNA, and/or RNA), polypeptides,peptidomimetics, fusion proteins or prodrugs thereof, antibodies,ribozymes, aptamers, small molecules, and other compounds that inhibitCAP adduct activity and/or formation. More specific examples of agentsthat inhibit CAP adduct activity and/or formation are disclosed in PCTPublication No. WO 2008/013797 A2, the entirety of which is herebyincorporated by reference.

Depending upon the type and severity of the pathological angiogenesis,the agents that substantially inhibit complexing of TLR2 with CAPadducts can be prepared as a pharmaceutical or therapeutic composition(described above) and then administered to the subject via anappropriate route (also described above). Upon administration to thesubject, the agent can substantially inhibit complexing of TLR2 with CAPadducts in endothelial cells. In the absence of TLR2 and CAP adductcomplexing, the TLR2 signaling pathway will not be activated and, inturn, will not lead to integrin expression by endothelial cells. Withoutan activated TLR2 signaling pathway, endothelial cells will notproliferate into the surrounding matrix to form solid sprouts connectingneighboring vessels. Consequently, pathological angiogenesis will besubstantially prevented or inhibited in the subject.

In accordance with another aspect of the application, the agents thatsubstantially inhibit complexing of TLR2 with CAP adducts may be used totreat animals and patients with aberrant angiogenesis, such as thatcontributing to a variety of diseases and disorders. The most prevalentand/or clinically important of these, outside the field of cancertreatment, include arthritis, rheumatoid arthritis, psoriasis,atherosclerosis, diabetic retinopathy, age-related macular degeneration,Grave's disease, vascular restenosis, including restenosis followingangioplasty, arteriovenous malformations (AVM), meningioma, hemangiomaand neovascular glaucoma. Other potential targets for interventioninclude angiofibroma, atherosclerotic plaques, conical graftneovascularization, hemophilic joints, hypertrophic scars, osler-webersyndrome, pyogenic granuloma retrolental fibroplasia, scleroderma,trachoma, vascular adhesions, synovitis, dermatitis, various otherinflammatory diseases and disorders, and even endometriosis. Furtherdiseases and disorders that are treatable by the composition described,and the unifying basis of such angiogenic disorders, are set forthbelow.

One disease in which angiogenesis is involved is rheumatoid arthritis,wherein the blood vessels in the synovial lining of the joints undergoangiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Factors associatedwith angiogenesis also have a role in osteoarthritis, contributing tothe destruction of the joint.

Another example of a disease mediated by angiogenesis is occularneovascular disease. This disease is characterized by invasion of newblood vessels into the structures of the eye, such as the retina orcornea. It is the most common cause of blindness and is involved inapproximately twenty eye diseases. In age-related macular degeneration,the associated visual problems are caused by an ingrowth of chorioidalcapillaries through defects in Bruch's membrane with proliferation offibrovascular tissue beneath the retinal pigment epithelium. Angiogenicdamage is also associated with diabetic retinopathy, retinopathy ofprematurity, corneal graft rejection, neovascular glaucoma andretrolental fibroplasia.

Other diseases associated with corneal neovascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis,syphilis, Mycobacteria infections, lipid degeneration, chemical bums,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, andconical graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagetsdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, Eales disease,Bechets disease, infections causing a retinitis or choroiditis, presumedocular histoplasmosis, Bests disease, myopia, optic pits, Stargartsdisease, pars planitis, chronic retinal detachment, hyperviscositysyndromes, toxoplasmosis, trauma and post-laser complications.

Other diseases include, but are not limited to, diseases associated withrubeosis and diseases caused by the abnormal proliferation offibrovascular or fibrous tissue including all forms of proliferativevitreoretinopathy.

Chronic inflammation also involves pathological angiogenesis. Suchdisease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells.

Another pathological role associated with angiogenesis is found inatherosclerosis. The plaques formed within the lumen of blood vesselshave been shown to have angiogenic stipulatory activity. Thisapplication provides an effective treatment for such conditions.

One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

Angiogenesis is also responsible for damage found in hereditary diseasessuch as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and may be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Diseases and disorders characterized by undesirable vascularpermeability can also be treated by the compositions described herein.These include edema associated with brain tumors, ascites associatedwith malignancies, Meigs' syndrome, lung inflammation, nephroticsyndrome, pericardial effusion and pleural effusion, as disclosed in WO98/16551, specifically incorporated herein by reference.

Each of the foregoing diseases and disorders, along with all types oftumors, as described in the following sections, can be effectivelytreated by the compositions described herein in accordance with theknowledge in the art, as disclosed in, e.g., U.S. Pat. No. 5,712,291(specifically incorporated herein by reference), that unified benefitsresult from the application of anti-angiogenic strategies to thetreatment of angiogenic diseases.

The agents that substantially inhibit complexing of TLR2 with CAPadducts can also be utilized in the treatment of neoplastic disorders,such as tumors or cancers. Tumors in which angiogenesis is importantinclude malignant tumors, and benign tumors, such as acoustic neuroma,neurofibroma, trachoma and pyogenic granulomas. Angiogenesis isparticularly prominent in solid tumor formation and metastasis. However,angiogenesis is also associated with blood-born tumors, such asleukemias, and various acute or chronic neoplastic diseases of the bonemarrow in which unrestrained proliferation of white blood cells occurs,usually accompanied by anemia, impaired blood clotting, and enlargementof the lymph nodes, liver, and spleen. Angiogenesis also plays a role inthe abnormalities in the bone marrow that give rise to leukemia-liketumors.

Angiogenesis is important in two stages of tumor metastasis. In thevascularization of the primary tumor, angiogenesis allows cells to enterthe blood stream and to circulate throughout the body. After tumor cellshave left the primary site, and have settled into the secondary,metastasis site, angiogenesis must occur before the new tumor can growand expand. Therefore, prevention of angiogenesis can prevent metastasisof tumors and contain the neoplastic growth at the primary site,allowing treatment by other therapeutics, particularly, therapeuticagent-targeting agent constructs.

The agents that substantially inhibit complexing of TLR2 with CAPadducts described herein are thus broadly applicable to the treatment ofany malignant tumor having a vascular component. In using the agentsthat substantially inhibit complexing of TLR2 with CAP adducts in thetreatment of tumors, particularly vascularized, malignant tumors, theagents may be used alone or in combination with, e.g., chemotherapeutic,radiotherapeutic, apoptopic, anti-angiogenic agents and/or immunotoxinsor coaguligands.

Typical vascularized tumors for treatment are the solid tumors,particularly carcinomas, which require a vascular component for theprovision of oxygen and nutrients. Exemplary solid tumors that may betreated include, but are not limited to, carcinomas of the lung, breast,ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas,small cell carcinomas, melanomas, gliomas, glioblastomas,neuroblastomas, and the like. WO 98/45331 is also incorporated herein byreference to further exemplify the variety of tumor types that may beeffectively treated using agents that substantially inhibit complexingof TLR2 with CAP adducts.

Knowledge of the role of angiogenesis in the maintenance and metastasisof tumors has led to a prognostic indicator for cancers, such as breastcancer. The amount of neovascularization found in the primary tumor wasdetermined by counting the microvessel density in the area of the mostintense neovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by the therapies described herein will reduce ornegate the recurrence of such tumors.

The compositions described herein can also be used in the treatment ofany patient that presents with a solid tumor. In light of the specificproperties of the agents that substantially inhibit complexing of TLR2with CAP adducts, the therapeutics described herein will have reducedside effects. Particular advantages will result in the maintenance orenhancement of host immune responses against the tumor, as mediated bymacrophages, and in the lack of adverse effects on bone tissue. Thecompositions will thus be the anti-angiogenic therapy of choice for thetreatment of pediatric cancers and patients having, or at risk fordeveloping, osteoporosis and other bone deficiencies.

Although all malignancies and solid tumors may be treated by thecompositions or agents described herein, the agents that substantiallyinhibit complexing of TLR2 with CAP adducts are particularlycontemplated for use in treating patients with more angiogenic tumors,or patients at risk for metastasis.

The compositions and methods described herein also intended as apreventative or prophylactic treatment. These aspects include theability of the invention to treat patients presenting with a primarytumor who may have metastatic tumors, or tumor cells in the earlierstages of metastatic tumor seeding. As an anti-angiogenic strategy, thecompositions and methods may also be used to prevent tumor developmentin subjects at moderate or high risk for developing a tumor, as basedupon prognostic tests and/or close relatives suffering from a hereditarycancer.

Therapeutically effective doses of the agents that substantially inhibitcomplexing of TLR2 with CAP adducts are readily determinable using datafrom an animal model. Experimental animals bearing solid tumors arefrequently used to optimize appropriate therapeutic doses prior totranslating to a clinical environment. Such models are known to be veryreliable in predicting effective anti-cancer strategies. For example,mice bearing solid tumors are widely used in pre-clinical testing.

In using the agents that substantially inhibit complexing of TLR2 withCAP adducts in anti-angiogenic therapies, one can also draw on otherpublished data in order to assist in the formulation of doses forclinical treatment. For instance, although the agents and methodsdescribed herein have distinct advantages over those in the art, theinformation in the literature concerning treatment with otherpolypeptides can still be used in combination with the data and teachingin the present application to design and/or optimize treatment protocolsand doses.

Any dose, or combined medicament of the agents that substantiallyinhibit complexing of TLR2 with CAP adducts, that results in anyconsistently detectable anti-angiogenic effect, inhibition ofmetastasis, tumor vasculature destruction, tumor thrombosis, necrosisand/or general anti-tumor effect. The application may also be effectiveagainst vessels downstream of the tumor, i.e., target at least a sub-setof the draining vessels, particularly as cytokines released from thetumor will be acting on these vessels, changing their antigenic profile.

It will also be appreciated that an agent that substantially inhibitscomplexing of TLR2 with CAP adducts in endothelial cells can be used incombination and adjunctive therapies for treating aberrant angiogenesis(e.g., neoplastic disorders). The phrase “combination therapy” canembrace the administration of the agent that substantially inhibitcomplexing of TLR2 with CAP adducts, and a therapeutic agent as part ofa specific treatment regimen intended to provide a beneficial effectfrom the co-action of these therapeutic agents. Administration of thesetherapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected). “Combination therapy” can embraceadministration of these therapeutic agents in a sequential manner; thatis, wherein each therapeutic agent is administered at a different time,as well as administration of these therapeutic agents, or at least twoof the therapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single dosage having a fixedratio of each therapeutic agent or in multiple, single dosages for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues.

The therapeutic agents can be administered by the same route or bydifferent routes. For example, a first therapeutic agent of thecombination selected may be administered by intravenous injection whilethe other therapeutic agents of the combination may be administeredorally. Alternatively, for example, all therapeutic agents may beadministered orally or all therapeutic agents may be administered byintravenous injection. The sequence in which the therapeutic agents areadministered is not narrowly critical. “Combination therapy” can alsoembrace the administration of the therapeutic agents as described abovein further combination with other biologically active ingredients (suchas, but not limited to, a second and different therapeutic agent) andnon-drug therapies (such as, but not limited to, surgery or radiationtreatment). Where the combination therapy further comprises radiationtreatment, the radiation treatment may be conducted at any suitable timeso long as a beneficial effect from the co-action of the combination ofthe therapeutic agents and radiation treatment is achieved. For example,in appropriate cases, the beneficial effect is still achieved when theradiation treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

The phrase “adjunctive therapy” can encompass treatment of a subjectwith agents that reduce or avoid side effects associated with thecombination therapy, including, but not limited to, those agents, forexample, that reduce the toxic effect of anticancer drugs, e.g., boneresorption inhibitors, cardioprotective agents; prevent or reduce theincidence of nausea and vomiting associated with chemotherapy,radiotherapy or operation; or reduce the incidence of infectionassociated with the administration of myelosuppressive anticancer drugs.

In one example, the therapeutic agent administered in combinationtherapy with the agents described herein can comprise anti-proliferativeagents. The phrase “anti-proliferative agent” can include agents thatexert antineoplastic, chmotherapeutic, antiviral, antimitotic,antitumorgenic, and/or immunotherapeutic effects, e.g., prevent thedevelopment, maturation, or spread of neoplastic cells, directly on thetumor cell, e.g., by cytostatic or cytocidal effects, and not indirectlythrough mechanisms, such as biological response modification. There arelarge numbers of anti-proliferative agent agents available in commercialuse, in clinical evaluation and in pre-clinical development, which couldbe used by combination drug chemotherapy. For convenience of discussion,anti-proliferative agents are classified into the following classes,subtypes and species: ACE inhibitors; alkylating agents; angiogenesisinhibitors; angiostatin; anthracyclines/DNA intercalators; anti-cancerantibiotics or antibiotic-type agents; antimetabolites; antimetastaticcompounds; asparaginases; bisphosphonates; cGMP phosphodiesteraseinhibitors; calcium carbonate; cyclooxygenase-2 inhibitors; DHAderivatives; DNA topoisomerase; endostatin; epipodophylotoxins;genistein; hormonal anticancer agents; hydrophilic bile acids (URSO);immunomodulators or immunological agents; integrin antagonists;interferon antagonists or agents; MMP inhibitors; miscellaneousantineoplastic agents; nitrosoureas; NSAIDs; ornithine decarboxylaseinhibitors; pBATTs; radio/chemo sensitizers/protectors; retinoids;selective inhibitors of proliferation and migration of endotheliaicells; selenium; stromelysin inhibitors; taxanes; vaccines; and vincaalkaloids.

The major categories that some anti-proliferative agents fall intoinclude antimetabolite agents, alkylating agents, antibiotic-typeagents, hormonal anticancer agents, immunological agents,interferon-type agents, and a category of miscellaneous antineoplasticagents. Some anti-proliferative agents operate through multiple orunknown mechanisms and can thus be classified into more than onecategory.

Another aspect of the application relates to a method for modulating oneor more inflammatory and/or autoimmune diseases or disorders mediated byTLR2 in a subject by administering to the subject a compositioncomprising an agent that modulates one or more CAP adducts in thesubject. Inflammatory and/or autoimmune diseases or disorders can referto diseases or disorders in which an activity of TLR2 in an immune cell(e.g., microglia, Schwann cells, monocytes, macrophages, dendriticcells, polymorphonuclear leukocytes (PMNs or PMLs), B cells (e.g., B1a,MZ B, B2), and T cells, including Tregs (e.g., CD4+CD25+ regulatory Tcells)), a signal transduction pathway of TLR2 in an immune cell, and/oran activity or signal transduction pathway that is mediated by TLR2 inan immune cell is involved. An inflammatory and/or autoimmune disease ordisorder mediated by TLR2 can include a disease or disorder where a TLR2activity or signal transduction pathway in an immune cell can bemodulated for treatment of the disease or disorder. Thus, theapplication can encompass compositions and methods that modulateinflammatory and/or autoimmune diseases or disorders mediated by TLR2activity in an immune cell and/or a TLR2 signal transduction pathway inan immune cell to provide a therapeutic benefit or therapeutic activityfor treatment of the disease or disorder.

Examples of inflammatory diseases or disorders that are mediated by TLR2and may be treatable can include, but are not limited to, airwayinflammation, asthma, autoimmune diseases or disorders, chronicinflammation, chronic prostatitis, glomerulonephritis, Behcet's disease,hypersensitivities, inflammatory bowel disease, reperfusion injury,rheumatoid arthritis, transplant rejection, ulcerative colitis, uveitis,conjunctivitis and vasculitis.

Examples of autoimmune diseases or disorders that are mediated by TLR2and may be treatable include, but are not limited to, lupuserythematosus, multiple sclerosis, type I diabetes mellitus, irritablebowel syndrome, Crohn's disease, rheumatoid arthritis, septic shock,alopecia universalis, acute disseminated encephalomyelitis, Addison'sdisease, ankylosing spondylitis, antiphospholipid antibody syndrome,autoimmune hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid,chagas disease, chronic obstructive pulmonary disease, coeliac disease,dermatomyositis, endometriosis, Goodpasture's syndrome, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativa,idiopathic thrombocytopenic purpura, interstitial cystitis, morphea,myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, perniciousanaemia, polymyositis, primary biliary cirrhosis, schizophrenia,Sjogren's syndrome, temporal arteritis, vasculitis, vitiligo, vulvodyniaand Wegener's granulomatosis.

The agent that modulates one or more CAP adducts in the subject caninclude any substance or molecule that promotes or inhibits one or moreCAP adducts. Prior to administration to the subject, one or more of theagents can be formulated into a pharmaceutical or therapeuticcomposition by compounding the agent with at least one pharmaceuticallyacceptable carrier. As discussed above, pharmaceutically acceptablecarriers are known in the art and may include any material or materials,which are not biologically or otherwise undesirable, i.e., the materialmay be incorporated or added into the composition without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components (i.e., the agents) of the composition.As also discussed above, the particular formulation, dosage, andadministration route of the agent may depend upon the subject'sindividual need, such as the particular inflammatory and/or autoimmunedisease or disorder from which the subject is suffering.

In one example, the agent that inhibits one or more CAP adducts caninclude any substance or molecule that inhibits the activity and/orformation (e.g., expression) of a CAP adduct, such as a CEP proteinadduct. For example, the agent can inhibit one or more biologicalactivities of a CAP adduct (e.g., a CEP protein adduct). An example of abiological activity of a CAP adduct is inflammation. The agent can bindto all or a portion of the CAP adduct (e.g., a portion of the CAPadduct, the CAP portion of the CAP adduct, or the adduct portion of theCAP adduct) under conditions in which the inflammatory activity of theCAP adduct is inhibited.

Examples of agents that can inhibit receptor-mediated effects of CAPadducts (e.g., CEP protein adducts) can include nucleic acids, fragmentsor derivatives thereof and vectors comprising such nucleic acids (e.g.,a nucleic acid molecule, cDNA, and/or RNA), polypeptides,peptidomimetics, fusion proteins or prodrugs thereof, antibodies,ribozymes, aptamers, small molecules, and other compounds that inhibitCAP adduct activity and/or formation.

In another aspect, a therapeutically effective amount of an agent thatinhibits CAP protein adduct activity and/or formation can beadministered to a subject suffering from an inflammatory disease, suchas Crohn's disease. Crohn's disease is an ongoing disorder that causesinflammation of the digestive tract. In subjects with Crohn's disease,it is believed that immune cells accumulate in the lining of theintestines, producing chronic inflammation, which leads to ulcerationsand bowel injury. The agent can include an antibody or fragment thereof(e.g., monoclonal or polyclonal) that binds to all or a portion of oneor more CAP protein adducts to inhibit CAP protein adduct activityand/or formation. The agent can be formulated into a pharmaceuticalcomposition and delivered to the subject via an appropriate route (e.g.,intravenous injection or direct administration into the bowel). Uponadministration to the subject, the agent can inhibit the formationand/or activity of CAP protein adducts by binding to all or a portion ofthe CAP protein adducts and thereby preventing TLR2:CAP protein adductcomplex formation. By inhibiting TLR2:CAP protein adduct complexformation, the TLR2 signaling pathway is not activated, which, in turn,does not lead to cytokine expression and thus chronic inflammation ofthe bowel.

Another aspect of the application can include a method for modulatingone or more inflammatory and/or autoimmune diseases or disordersmediated by TLR2 in a subject by administering to the subject acomposition comprising an agent that promotes TLR2 activation in animmune cell of the subject. Agents that promote TLR2 activation caninclude TLR2 agonists. As described above, TLR2 agonists can generallyinclude any substance or molecule that binds to TLR2 present on immunecells and induces a response in the immune cells. For example, bindingof a TLR2 agonist to TLR2 can promote cytokine expression (e.g., tumornecrosis factors and interleukins) in an immune cell. Examples of TLR2agonists can include CAP adducts (e.g., CEP protein adducts) andPam₃CSK₄.

In one example of the application, a therapeutically effective amount ofa TLR2 agonist (e.g., a CEP protein adduct) can be administered tosubject suffering from a bacterial infection. At least one CEP proteinadduct can be formulated into a pharmaceutical composition and thendelivered to the subject via an appropriate route (e.g., intravenousadministration). Upon administration to the subject, a CEP proteinadduct can bind to TLR2 on immune cells and thereby activate the TLR2signaling pathway. Activation of the TLR2 signaling pathway can promoteexpression of various cytokines by the immune cells (e.g., tumornecrosis factors and interleukins) and lead to an increased inflammatoryresponse (e.g., fever) in the subject, which can subsequently promoteclearance of the bacterial infection.

Another aspect of the application relates to a method for treating adermatological disorder in a subject by administering a therapeuticallyeffective amount of a composition comprising an agent that promotes orinhibits TLR2 activation. Dermatological disorders can include anydisorder of skin, hair, or glands. A dermatological disorder can bemanifest in the form of visible lesions, pre-emergent lesions, pain,sensitivity to touch, irritation, inflammation, or the like.Dermatological disorders can include disorders of the cutaneous andpilosebaceous unit or the process of keratogenesis. For example, adermatological disorder can be a disorder of the epidermis or dermis, orwithin and surrounding a pilosebaceous unit, which is located within theepidermis, dermis, subcutaneous layer, or a combination thereof.Examples of dermatological disorders include, but are not limited to,acne, alopecia, psoriasis, seborrhea, ingrown hairs andpseudofolliculitis barbae, hyperpigmented skin, cutaneous infections,lichen planus, Graham Little Syndrome, periorificial dermatitis,rosacea, hidradenitis suppurativa, dissecting cellulitis, systemic lupuserythematosus, discoid lupus erythematosus, and the like.

Agents that promote TLR2 activation are described above and can includeTLR2 agonists, for example, a CAP adduct, such as a CEP adduct.

Agents that inhibit TLR2 activation are also described above and caninclude, for example, TLR2 antagonists, such as TLR2 antibodies, or anagent that inhibits one or more CAP adducts.

Depending upon the particular dermatological disorder, the agent thatpromotes or inhibits TLR2 activation can be formulated into apharmaceutical or therapeutic composition by compounding the agent withat least one pharmaceutically acceptable carrier. As discussed above,pharmaceutically acceptable carriers are known in the art and caninclude any material or materials which are not biologically orotherwise undesirable, i.e., the material may be incorporated or addedinto the composition without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other components(i.e., the agents) of the composition. As also discussed above, theparticular formulation, dosage, and administration route of the agentmay depend upon the subject's individual need, such as the particulardermatological disorder from which the subject is suffering.

One example of the application can include a method for treating adermatological disorder, such as alopecia in a subject by administeringto follicle cells of the subject a therapeutically effective amount of aTLR2 agonist that promotes hair growth in the subject. “Alopecia” canrefer to partial or full baldness, hair loss, and/or hair thinning, aswell as primary cicatricial alopecia (CA), which can refer to a group ofhair disorders that cause permanent destruction of the hair follicle.CAs can be classified as lymphocytic, neutrophilic, and combinationsthereof (i.e., “mixed”). Examples of lymphocytic CAs can include lichenplanopilaris, frontal fibrosing alopecia, chronic cutaneous lupus,erythematosus, pseudopelade, central centrifugal alopecia, alopeciamucinosa, and keratosis follicularis spinulosadecalvans. Examples ofneutrophilic CAs can include folliculitis decalvans, tuftedfolliculitis, and dissecting cellulitis. Examples of mixed CAs caninclude follicullitis keloidalis and erosive dermatosis.

In a subject suffering from alopecia (e.g., baldness), a TLR2 agonist,such as a CAP adduct can be formulated into a topical formulation. Forexample, the CAP adduct can be formulated into an ointment, cream, gel,paste, and/or oleaginous or water-soluble ointment base. Afterformulating the CEP adduct, a therapeutically effective amount of thetopical formulation can be applied to the scalp of the subject. Asillustrated in FIGS. 12-15, the number of hair follicles is increasedupon application of CAP adducts. Administration of a therapeuticallyeffective amount of a pharmaceutical or therapeutic compositioncomprising a CAP adduct to a subject suffering from alopecia (e.g.,baldness) can thus lead to an increase in the number of hair folliclesand thereby promote hair growth.

The following example is for the purpose of illustration only and is notintended to limit the scope of the claims, which are appended hereto.

Example 1

Materials and Methods

Animal Studies

Wild type C57BL/6 and DsRed transgenic mice were obtained from JacksonLaboratory, CD36^(−/−) from Dr. M. Febbraio (Cleveland Clinic),SR-B1^(−/−) from Dr. M. Kruger, MyD88^(−/−) and TLR2^(−/−) from Dr. S.Akira (Osaka University). All mice were maintained in the BiologicalResources Unit of the Lerner Research Institute, accredited by theAssociation for the Assessment and Accreditation of Laboratory AnimalCare International. All experimental work was conducted in compliancewith the Institutional Animal Care and Use Committee program.

Immunohistochemistry and Image Analysis

For immunofluorescence staining, we used rabbit polyclonal anti-CEP(described in Crabb, J. W. et al., Proc Natl Acad Sci USA99(23):14682-14687, 2002), anti-CD31 (BioLegend), anti-αSMA (Rockland).We fixed 7 μm thick tissue sections with 4% paraformaldehyde andembedded in paraffin or in OCT freezing medium. After incubation withprimary antibodies, samples were washed with PBS and exposed to a AlexaFluor-labeled secondary antibody, either goat anti-rabbit AlexaFluor488, anti-rat Alexa Fluor568, or anti-mouse Alexa Fluor568(Invitrogen). The slides were mounted with medium (DakoCytomation) andimages were taken by either a TCS-SP (Leica) or a ZMZ1000 (Nikon)microscope. For quantification, the images were analyzed with ImageProsoftware (MediaCybernetics).

Bone Marrow Transplant (BMT) and Wound Assay

We performed BMT as previously described (Chen et al., Nat Med.11(11):1188-1196, 2005). Briefly, we subjected two month old male wildtype or TLR2^(−/−) mice to irradiation with a total dose of 9 Gyfollowed by bone marrow reconstitution by tail vein injection with 10⁷bone marrow cells isolated from donor femurs. 8 weeks after BMT, micewere used for wound healing assays. A back punch wound healing model wasused as described elsewhere (Feng, W. et al., J Cell Biol.183(6):1145-1157, 2008). Mice were given PBS or CEP-dipeptide peritonealinjections (1.4 μg per mg of body weight) at the time of injury and ondays 2, 4, and 6.

Aortic Ring Assay

The mouse aortic ring assay was performed as previously described(Mahabeleshwar, G. H. et al., Methods Mol Med. 129:197-208, 2006).

Isolation of Endothelial Cells

Mouse lungs were excised, minced, and digested using acollagenase-dispase reagent (Roche Diagnostic). Digests were strainedand the resulting cell suspension was plated on flasks coated with 1mg/ml fibronectin. Thereafter, endothelial cells were isolated andcharacterized as described previously (Mahabeleshwar, G. H. et al.,Methods Mol Med. 129:197-208, 2006).

Cell Adhesion

We performed cell adhesion assays as described elsewhere (Byzova, T. V.et al., J Cell Biol. 143(7):2081-2092, 1998). Briefly, 10⁴ HUVEC or MLECcells were added to ligand coated wells of a 96-well plate and incubatedfor 1 h in the presence of CEP-adduct or VEGF as indicated. The cellswere washed with and fixed with 4% paraformaldehyde in PBS. Thereaftercells were contrasted by hematoxylin staining and photographs weretaken.

Cell Migration Assay

We used fibronectin-coated transwell inserts of 8 μm pore size(Corning). HUVEC or MLEC cells were added into each well. The lowerchamber was complemented with an attractant as indicated. Cells wereallowed to migrate for 5 h and fixed with 4% paraformaldehyde for 20 minthen stained with hematoxylin. The photographs of 4 random fields weretaken using a phase contrast inverted microscope (Athena).

Tube Formation Assay

HUVEC or MLEC cells were seeded on Matrigel-coated plates (BDBioscience). Medium was supplemented with VEGF or protein adducts asindicated, and cells were further incubated at 37° C. for 8 h. Tubeformation was observed using a phase contrast inverted microscope, andphotographs were taken from each well. The data were quantified bymeasuring the length of tubes with ImagePro software.

Hind Limb Ischemia Model

The femoral artery was ligated near the caudally branching deep femoralartery and a second ligation was placed in proximity of the tibialarteries branching. The portion of the artery and vein between theligation points was excised. Hind-limb blood flow was measured by alaser Doppler moorLDI2-IR near infrared laser Class 3R (MoorInstruments) in arbitrary perfusion units (PU). The results were plottedas a ratio of surgery to nonsurgery leg for each animal to account forvariations between animals.

Rac1 Activation Assay

Per assay, we used 2×10⁶ cells lysed in 500 μl buffer: 25 mM HEPS KOH pH7.5; 250 mM NaCl, 1% NP-40, 10% glycerol, 10 mM MgCl₂ 2 mM EGTA.GST-PAK-PBD pre-absorbed on glutathione agarose beads (Cytoskeleton) wasadded in the amount of 20 μl 50% slurry followed by incubation on arotary shaker for 45 min at 4° C. Beads were washed four times withlysis buffer before elution with SDS-PAG sample buffer. Rac1 wasdetected by Western Blot (clone 102, BD Bioscience).

Luciferase Reported Assay

We transfected HEK 293 cells by hTLR2 expression plasmid (InvivoGen) orcontrol vector, as well as NF-kB luciferase reporter (gift from Dr. P.Chumakov, Cleveland Clinic). 24 hrs post-transfection cells were exposedto CEP adduct or the carrier protein for an additional 8 hrs. Reporteractivity test was performed with a luciferase assay system (Promega) andthe readout was normalized to the protein amount.

ELISA

We immobilized 2 μg CEP-KLH or KLH on a 96-well polystyrene plate(Thermo Labsystems) overnight at 4° C. and blocked with 5 mg/ml BSA onPBS, followed by a wash with 1 mg/ml BSA. We added the indicated amountsof recombinant TLR2 extracellular domain (R&D Systems) for 4 h at roomtemperature then washed the plates. Detection was done with anti-TLR2antibodies (clone TL2.1, eBioscience) and anti-mouse HRP-coupledantibodies (Bio-Rad).); quantitation was done by a colorimetric assay(R&D Systems) at 560 nm using a Vmax plate reader (Molecular Devices).

Statistical Analysis

All data are presented as mean±s.e.m. for all studies. Probabilityvalues were based on the paired t test: NS—not significant, *P<0.05, **P<0.01, *** P<0.001.

Results/Discussion

As shown in FIG. 1, the adducts, CEP in particular, can be detectedduring the wound healing process in a transient fashion. The levels ofCEP increase dramatically in 5 day old wounds compared to normal skinand return to their original levels when the wound is completely healed(in 28 days) (FIG. 1A). This increase coincides with the recruitment ofbone marrow derived cells (FIG. 1B), which contribute to oxidation bymeans of respiratory burst (Segal, A. W. Annu Rev Immunol. 23:197-223,2005). Based on co-staining, it appears that F4/80+ macrophages but notGr-1+ neutrophils accumulate CEP (FIG. 1C). Importantly, high levels ofCEP are apparent at the stage of intense wound vascularization asevidenced by CD31 and CEP co-staining, suggesting a possible role forthis adduct in wound angiogenesis (FIG. 1A). In contrast to thetransient presence of CEP during the wound healing process, its levelswere continuously high in pathological specimens. In a mouse model ofmelanoma, a tumor characterized by excessive vascularization with asubstantial inflammatory component, intensity of CEP staining is ˜9times higher than in normal uninjured skin (FIG. 1D). Likewise, CEP waspresent at higher levels in human melanoma specimens compared to normalskin from the same donor (FIG. 1E). In uninjured muscle tissue, CEP isoften present within the vascular wall and is colocalized with the SMAlayer of arterioles (FIG. 1F). Remarkably, the intensity of CEP stainingincreased substantially with aging (FIG. 1G), possibly reflectingaccumulation of oxidative products over time. Taken together, theseresults suggest that the end products of lipid oxidation, represented byCEP might serve as important regulators of inflammation-associatedvascularization.

Indeed, as shown in FIG. 2, CEP directly promotes angiogenic responsesof isolated endothelial cells of various origins. When tested on HUVEC,mouse lung (MLEC), or mouse aortic endothelial cells (MAEC), thepro-angiogenic effect of CEP was comparable to that of VEGF, as assessedby tube formation (FIGS. 2A-B), aortic ring (FIG. 2C) and cell migrationassays (FIG. 2D). The protein moiety did not influence CEP's effect, asadducts coupled to BSA, HSA or a dipeptide were equally effective inpromoting angiogenic responses (FIGS. 2A-D). Similar to VEGF, the effectof CEP was integrin dependent (FIG. 6A). However, in contrast to VEGF,stimulation of endothelial cells with CEP did not result in VEGFR2phosphorylation (FIG. 6B). Thus, CEP is able to activate proangiogenicresponses of isolated endothelial cells by a mechanism independent ofVEGF/VEGFR2 signaling. Importantly, adducts from the same family of CAP(FIG. 5) represented by CPP (carboxypropylpyrrole) were alsopro-angiogenic (FIGS. 2A-H), implying that the EC recognize and respondto the structural pattern rather than to a particular chemical moiety.

To address the nature of the possible receptor mediating CEP-inducedangiogenesis, we considered the role of scavenger receptors which arecapable of pattern recognition. We assessed angiogenic responses to CEPand CPP adducts using EC from CD36^(−/−) and scavenger receptor B1(SR-B1)^(−/−) mice. Both adducts promoted endothelial sprouting (FIG.2E) and tube formation (FIG. 2F) of CD36^(−/−) EC and were as effectivesimulators as VEGF. Likewise, ablation of the functionally similar SR-B1receptor had no substantial impact on EC responses to CEP and CPP (FIGS.2G-H) indicating that scavenger receptors are not involved inrecognition of these adducts.

Since EC appear to respond to CAPs molecular pattern and the presence ofthis pattern is a characteristic of oxidative stress, we hypothesizedthe involvement of Toll like family receptors (TLRs). Of all TLRs, wefocused on TLR2 and TLR4, since they are expressed on endothelium andknown to recognize a broad range of pathogen-derived and endogenousligands, both of lipid and protein nature (Zahringer, U. et al.,Immunobiology 213(3-4):205-224, 2008). As shown in FIG. 3A, blockingantibodies against TLR2 but not TLR4 specifically inhibit CEP—but notVEGF-induced endothelial tube formation. Similar results were observedin EC migration assays (FIG. 7A). To further address the role of TLR2 inCEP-driven angiogenesis, EC from TLR2^(−/−) mice were compared to thosefrom TLR2^(+/+) controls. As shown in FIG. 3, TLR2^(−/−) EC did notrespond to CEP or CPP treatment in either tube formation or aortic ringsprouting assays (FIGS. 3B and 3C, respectively). At the same time,VEGF-triggered EC responses were not affected by the lack of TLR2 (FIGS.3B-C). Similar results were observed in cell migration assays (FIG. 7B).To confirm the key role of TLR2 in pro-angiogenic responses of EC, theTLR2 synthetic ligand Pam3CSK4 (Aliprantis, A. O. et al., Science285(5428):736-739, 1999) was tested under similar conditions. The ligandinduced robust sprouting of EC from aortic rings of TLR2^(+/+) but notTLR2^(−/−) mice (FIG. 3D). Likewise, in two other functional assays, ECtube formation and EC adhesion, Pam3CSK4 was as effective a stimulatoras CEP or VEGF. The blockade of TLR2 using antibodies inhibited theeffect of Pam3CSK4, but not VEGF (FIGS. 7c -D). Therefore, activation ofendothelial TLR2 by CEP or by its synthetic ligand promotes angiogenicresponses which are VEGF independent.

Having established the role of TLR2 in the proangiogenic effect of CEPin vitro, we considered the possibility that exogenous CEP might promotevascularization in vivo. In the hind limb ischemia model, CEP injectionsenhanced tissue revascularization after ligation surgery. As shown inFIG. 3E, CEP stimulated an increase in vascular density, as assessed bythe number of SMA-positive vessels per field, as well as an increase inaverage vascular area. These proangiogenic effects were dependent onTLR2 since TLR2^(−/−) mice were not responsive to exogenous CEP andexhibited substantially impaired revascularization (FIG. 3E). As aconsequence, CEP injection resulted in the stimulation of blood flow inTLR2^(+/+) but not TLR2^(−/−) mice (FIG. 3F). The enhanced blood flowwas due to increased angiogenesis (FIG. 3E) and, possibly, to therestructuring of collateral blood vessels bypassing the ligation of thefemoral artery (FIG. 8).

Since CEP synthesis is a common phenomenon in wound healing (FIGS.1A-C), we addressed the role of this adduct in wound vascularization. Todistinguish the effect of CEP on EC from that on inflammatory cells,TLR2^(−/−) mice were transplanted with TLR2^(+/+) bone marrow. Theresulting TLR2^(+/+)>TLR2^(−/−) and TLR2^(+/+)>TLR2^(+/+) chimeras wereused for wound assays. CEP injection into TLR2^(+/+)>TLR2^(+/+) animalsresulted in augmented vascularization and more than three-fold fasterwound closure when compared to the PBS-treated group (FIGS. 4A-B). Atthe same time, the wounds of TLR2^(+/+)>TLR2^(−/−) animals healedsubstantially slower, and CEP had no effect on either wound closure orvascularization in these mice (FIGS. 4A-E). Quantification of thevasculature in the wounded tissue revealed a 2.5-fold increase invascular area and a 1.7-fold increase in vascular density resulting fromCEP treatment compared to PBS treatment (FIG. 4C). The lack of TLR2 onnon-hematopoietic cells completely abrogated proangiogenic activity ofCEP (FIG. 4C), indicating the key role for this receptor. Together withthe results of experiments on isolated endothelial cells, these datashow that TLR2 on vascular but not on immune cells is responsible forrecognition of the end-products of lipid oxidation and initiation ofproangiogenic signaling.

The key mechanism underlying broad ligand specificity of TLR2(Zahringer, U. et al., Immunobiology 213(3-4):205-224, 2008) is itsheterodimerization with other members of the TLR family (Ozinsky, A. etal., Proc Natl Acad Sci USA 97(25):13766-13771, 2000) and its ability toform complexes with heterologous co-receptors (Hoebe, K. et al., Nature433(7025):523-527, 2005). Thus, it is possible that recognition ofoxidized lipids might require a co-receptor for TLR2. However, whentested in ELISA, recombinant TLR2 bound the CEP protein adduct, but notthe carrier protein alone, indicating the possibility of a directinteraction between CEP and TLR2 (FIG. 9A). Importantly, similar toother established ligands for TLR2, CEP triggers a major TLR signalingevent, NF-kB activation. As shown in NF-kB luciferase reporter assay,CEP induced NF-kB activation and TLR2 was required for this response(FIG. 9B).

Next, we considered whether MyD88 adapter protein, a known mediator ofTLR2 signaling, is involved in proangiogenic activity of CEP. As shownin FIG. 4D, CEP-induced EC sprouting is MyD88-dependent sinceMyD88^(−/−) cells did not respond to stimulation by CEP. At the sametime, VEGF-induced angiogenesis was not affected by the lack of MyD88(FIG. 4D). Considering that CEP-induced angiogenic responses areintegrin dependent, we focused on mediators common for integrin and TLRsignaling. Rac1 small G protein is a key regulator of integrin-mediatedmigration (Tan, W. et al., FASEB J. 22(6):1829-1838, 2008), which isknown to function in vascular development, and, most interestingly, isreported to be activated downstream of TLR (Arbibe, L. et al., NatImmunol. 1(6):533-540, 2000). Accordingly, we assessed Rac1's GTP loadin response to CEP treatment as a measure of pro-migratory signalinginduced by the compound. As shown in FIG. 4E, Rac1's GTP-bound form isreadily detected after CEP treatment of TLR2^(+/+), but not TLR2^(−/−)or MyD88^(−/−) cells. Thus, lipid oxidation products, represented byCEP, promote angiogenic responses of EC by activating the TLR2 signalingpathway in a MyD88-dependent manner, leading to Rac1 activation which,in turn, facilitates integrin function.

These findings demonstrate the presence of a novel mechanism ofangiogenesis which is independent of hypoxia-triggered VEGF expression.In this model, the products of lipid oxidation are generated as aconsequence of inflammation, recruitment of myeloid cells, andrespiratory burst. These products are directly recognized by TLR2 on ECand promote angiogenesis in vivo, thereby contributing to acceleratedwound healing and tissue recovery after ischemic injury. However, ifhigh levels of CEP and its analogs are accumulated in tissues, it mightlead to excessive and pathological vascularization, e.g., in tumors.

Example 2

In this example, as illustrated in FIG. 12-15, we show the effect of CEPdipeptide on hair growth is TLR2 dependent. Petrolatum (1 g) was mixedwith CEP-dipeptide (150 microg) for 10 min. Wounds (6 mm in diameter)were created and petrolatum was applied every day (˜1 mg of mixture tocover the wound area) for 2 weeks. The reason to apply it every day isthat mice roll in their bedding and remove any ointments applied. Higherconcentration of 300 microg CEP per 1 g of petroleum was found to havean effect similar to that of 150 microg/g. Concentrations higher than300 microg CEP per 1 g of petroleum were less effective than 150-300microg CEP per 1 g. FIG. 12 A shows representative images of WT and TLR2null mice treated with petroleum with or without CEP. FIG. 12B shows H&Estained sections of skin showing hair follicles of WT and TLR2 null micetreated with or without CEP.

FIG. 13 illustrates quantitative results showing increased density ofhair follicles upon CEP application in TLR2^(+/+) but not TLR2^(−/−)mice as described above. Please note that hair follicles are known toexpress TLR2 as a part of immune defense.

FIGS. 14-15 illustrate theame experiment using bone marrow chimeras ofTLR2^(+/+) and TLR2^(−/−) mice. The purpose of this experiment is toshow that effect of CEP is not bone marrow (inflammation) dependent.

From the above description, those skilled in the art will perceiveimprovements, changes and modifications. Such improvements, changes, andmodifications are within the skill of those in the art and are intendedto be covered by the appended claims. All patents, publications, andreferences recited herein are incorporated by reference in theirentirety.

Having described the invention, the following is claimed:
 1. A method ofpromoting hair growth of a mammalian subject, comprising: administeringto follicle cells of the mammalian subject a therapeutically effectiveamount of a TLR2 agonist that promotes TLR2 activation and hair growthof the mammalian subject, the TLR2 agonist comprising acarboxyethylpyrrole (CEP) adduct having the following formula:

wherein X is a carrier molecule, the carrier molecule selected from thegroup consisting of a dipeptide, mouse serum albumin and human serumalbumin, and is bound to the amine of the CEP pyrrole orpharmaceutically acceptable salts thereof.
 2. The method of claim 1,wherein the therapeutically effective amount is the amount required toincrease hair follicle density of the subject.